Inhibition of isoniazid acetylation



numrnon on ISONIAZIE) ACETYLATION Willard 3. Johnson, Montreal, Quebec, Canada, assignor to Frank W. Homer, Limited, Montreal, Quebec, Elana'dz, a corporation of Canada No Drawing. and Dec. s, 1956, S81. No. 625,522 Claims priority, application Canada M23130, 1956 e 'Cia'ims. Cl. 167-65) This invention relates to a therapeutic composition and more particularly to a therapeutic composition including isoniazid and an acetyla'tion inhibitor.

In applicants copending United States application 452,227, filed August 25, 1954, now abandoned,- for an invention of Willard Johnson entitled Therapeutic Composition Including Acetylation Inhibitor of which this application is a continuation in part, it was explained that the acetylation of sulfOnamides with consequentinf activation and loss of the sulfonamides and possibilities of kidney damage could be inhibited by administering with the sulfona rnides a substance which would compete with the sulfdnarnides for the acetylating enzyme. The inhibition this caused tends to reduce the danger of kidney damage and, by reducing the rate of urinary excretion makes possible the maintenance of therapeutic blood levels of free sulfonarnide at lower dosage levels and with less frequent administration than was hitherto the case. It was also disclosed thatthe acetylation inhibitors inhihited'the acetylation of isoniazid.

The inhibition of the acetylationof isoniazid rovides" nited States l liatent "O a means of attaininggreatereliectiveness-of a given dose of isoniazidby the use of inhibitors of isoniazid acetylation to prevent the inactivation and rapid excretionof the drug. Theinhibitors ofisoniazidacetylation are-able to produce elevated and sustained blood plasma levels of free, therapeutically active isoiiiaiid, thus obviating the necessity of giving increased dosages to obtain higher blood levels of isoniazid. The instant application fur, ther develops the use of "applicants invention in the acetylatidn ofisonia'zid '7 isoniazid, as an anti-microbial drug, is consideredto be equivalent if not superior to streptomycin in the treatment of pulmonary tuberculosis. However it has been shown by Hughes, J. Pharmacol. & Exper. Therein, 109i 7 44 (1935},aridcdnfirrhed by others; Nature, l73: 35

{1954), that when isoniazid is administered to and monkey, from to of the drug appears in the urine in the acetylated form, that is, as 1-isonicotinyl-2- acetylhydrazine.

Acetyl-isoiiiazid has less than one five-hundi edth'the.

. 2 has been reached. "The over-all effect ofac'etylatidn is in all probability a diminution in the therapeutic effectiveness of the. administered drug.

Both isoniazid and su-ltonamides are act'yla'ted" by'th'e same enzyme'sys'tem which is located in the liver of birds and mammals (including man {The dual ac etylation of isoniazid and sul fan ilamide is'illustratedby the experiment'showii in Table 'Im-lsoniazid and sulfanilamide, separately and in combination, were incubated with a cell-free extract-ofpigeon liver under conditions suitable for acetylation' to take place. When-2.92 micromoles of isoniazid and 2.61 micrornoles of sulfanilamide were incubated separately with the liver extract, 1-. 51 micromolesof'isoniazid'and 0.9 0 micremcle of s'ul'fanilarnide were acetylated, When the same amounts of isoniazid and sulfanilam-ide were incubated in combination, only 1.05 micromolesof'isonia'zid and 0.30 microrfiole of sulfanilamide were acetylated; that is, a reaucti ir in acetylation of 30% and 68% respectively. This indicates that isoniazid and sulfanilarnide compete for the same enzyme. Therefore, inhibitors of sulranilamide acetylation will inhibit, also, the acetylation of isoniazid.

TABLE I J NH Inhibition: Sulfa- Inhibition at'etyof INl-I nilaniide of sulianil- Additions lated, acetylaacetylated, arrido M. tion, ,M acetylation,

Percent Percent INH 'Sulfanilamidm INH, Sulfanilamide--.

Vessel components: l-rnl-.-ot pigeon liver extract; 60

, ,uM potassiumphosphate.butfer (,pH7.4); 60 ,uM acetate;

- are soluble, non-toxic compounds selected from the group having the general formula where R is at least one member selected from the group consisting of hydrogen, halogen, hydroxyl, amino, lower alkyl radicals having not more than three carbon atoms,

: phenyl, and carboxyl, and where A is selected from the] group consisting of pyridine, benzamide, benzoic 'acidhydrazide, and benzoylhydroxamic acid, and pharactivity of free isoniazid against the tubercle bacillus in vitro, while in vivo the parent drug is at least times more active than the acetyl derivative (3). Moreover, as is known to be the case with sulr'onamides, the acetylderivative is excreted via the kidney at a much greater rate than is the free drug. Thus, acetylation of isoniazid by the liver leads to rapid inactivation and excretion of the drug, which to some extent accounts for the rapid drop in isoniazid blood levels after the peak blood level merits "'a'rerepio'diiced below "Table II.

'maceu'tlically, acceptable salts thereof.

Acell-free extract of pigeon liver was employed for testing the inhibitory elfect of various compounds on isoniazid acetylation. The compounds which were shown to be particularlyefiective werep-amino'salic'ylamide, N ,N -diethylsulfanilamide and gentisamide,:;1-

amindsalicyclic acid is also, as shown by experiments with rabbits, an 'e'ifective acetylation inhibitor when used in the ratio of A to 5 of isoniazidto para-amihosalicylic acid. There'su'lts obtained in the in'vitrozexpri- 3 TABLE 11 4 nhibition of isoniazid (INH) acetylation in vitro of free isoniazid were determined at intervals of 1 A, 2 A and 4 hours after dosage. In both rabbits the ad- 1 Pigeon liver acetylating system (see W. Johnson, Canad. .l. Biochcm. Tho IN H concentration was 8X10-4M. unless 1 In these experiments the IN?! concentration was 7.75XIO M. INH concentration in this experiment: 9.7Xl'tM.

Physloi. 33:107 1955). otherwise indicated.

Eflec! of p-amino plasma levels of free isoniazi lowing a single oral dose TABLE III Concentration Percent Inhibitor of liilllhilOl, iDhihlLlOD. the plasma levcl of {re moles/litre ol acetylatlon 5-Bromosalicylan1ide 1 3 ill- 52 Balicylamide g; 10

58 o-Oresotamide a 36 Gentisamlde g: p-hydroxybenzamide 2 ill 22 Gallic Acid Amide 8x10 29 p-Aminosalicylic Acid il 49 p-Aminosalicylanlirie 10- 86 o-Hydroxyhenzalisonicotiuyl hydrazoue" 5 70 p-Aminobcnzoic Acid.. 10- 41 B-Aminonlcotinamide 10- 50 p-Aminophcnylacetic Acid ZXiO- 36 4-Amino-5-imidazolecarboxamido 31 Pyrazinamide 1 19 20 NLN -Diothylsulianilamide $1 sulfamethylthiadlazole Z1 Suilame 16 Sulfacetamide l3 Sullanilamide 3 30 l-Hydrazinophthalazine 2x10 53 ministration of PAS with isoniazid resulted in an enhanced plasma level of tree isoniazid. This effect of PAS was most striking at the four-hour interval, when e isoniazid was more than twofold that of the control level. A similar efiect of PAS is shown inExperiment 2 of Table 111.

It should be mentioned that 24 hours after the administration of a single dose of isoniazid there' is no detectable sign of the drug in the bloodstream, and hence no carry-over of isoniazid from one experiment to the next. As an extra precaution the order of drug administration was reversed; that is, isoniazid alone was given 48 hours after the dose of isoniazid plus PAS. 7 Experiments were performed to ascertain the relationship between the dose level of PAS and the plasma level of free isoniazid. The same three. rabbits were used throughout this set of experiments. The isoniazid dose was kept constant at 50 mg. per kg. body weight and the sodium PAS dose was varied from 0 to 600 mg. per kg. body weight. That is, the isoniazid to sodium PAS ratio, on a weight basis, varied from 1:0 to 1: 12. Typical results are shown in Table IV. It can be seen that there is a progressive increase in the plasma level of free isoniazid at the 1% hr. and 4 hr. intervals as the dose of sodium PAS is increased. Thus, when isoniazid and sodium PAS were administered in the ratio of 1:12 the plasma level of free isoniazid was 209% of the level attained when isoniazid alone was given.

salicyclic acid (sodium) on blood d (INH) in rabbits fol- Blood Plasma Levels of Free Isoniazid.

Expt. Rabbit Weight, Drug Dose (mgJkg. body wt.) 1% hours 2% hours 4 hours No. No. kg.

mg., Percent mgz. Percent mg.. Percent percent 01 control percent of control percent of control 7 1 6 jCnntrollINTi- 4.8 2 4 1. 4 1 llNIi50+PAS (Sodium) 500--. 6. 3 131 5 2 217 2.9 207 2 6 ]Cnntrol (Nil-I50 3.1 2 1 1.1

lIN'll5(l+PAS (Sodium) 500- 4. 9 126 3 4 162 2. 4 218 3 3 {Control (INHEO) 1. 58 0.22 2 .lNlIHH-PAS (Sodium) rIOlL 2.76 111 1.4 636 4 3 ,Control (1.\lI50) 2. 24 0 6 IINH50+PAS (Sodium) r100--- 2. S6 128 1. 86 290 7 TABLE IV Table HI shows '2 experiments in'which sodium PAS was employed as an inhibitor of isoniazid .acetylation. In the first experiment 2 rabbits, each weighing 6 kg.,' were given 300 mg. of isoniazid by stomach tube. Forty eight hours later the same rabbits were given 300 mg.;of isoniazid plus 3 g. of.soc1iul7ti PAS .v Blood plasma levels 1. In

Efiect 0] increasing doses of sodium PAS on the blood P10371211 levels 0] free isoniazid in rabbits j 'The same 3 rabbits, each weigh-rig 3 kg were used foresciy exnerimcnt.

I Free isoniazid blood plasma levels as shown are average valu s for the group of 3rabbits It has been verified by clinical tests on tuberculous patients that in 23 of 25 patients who received both isoniazid and sodium paraaminosalicylate a detectable increase in isoniazid blood level occurred (Mandel et al., Proc. Soc. Exp. Biol. andMecL, 9-1, 409, 1956).

order to develop the tulladvantages of this invention where the acetylation inhibitor is p-aminosalicylamide, a ratio of isoniazid to p-aminosalicylamide of between 1:5 and 1:15 should be maintained. A suitable dose for an adult would be a tablet containing as its active ingredients 50 mg. isoniazid and 500 mg. p-aminosalicylamide, to be administered several times daily.

Table V which is based on data reported by Mandel et al. gives the results of clinical testing of the invention and demonstrates its safety and effectiveness.

4. A therapeutic composition comprising as its active ingredients isoniazid and p-aminosalicylamide in a ratio of between 1:5 and 1:15.

5. A therapeutic composition comprising as its active ingredients isoniazid and gentisamide.

6. A therapeutic composition comprising as its active ingredients isoniazid and a substance selected from the group consisting of P-aminobenzoic acid and the nontoxic salts thereof.

TABLE V Antimicrobially active isoniazid Time of determi- Subject+amount of nation after test Serum level acetylation inhibitor dose of isoniazid given with test dose of 4 mg. per kg.

and 3 hours later body weight INH INH+ IN H+ IN H+ alone, PAS, PABA, PAS- ng. per g. per g. per amide mg. mg. mg.

3 0. 8 1. 6 3. 2 1 mi) {a 0.4 0.8 1.6 2 (2.5 ms.) 3;; 2 3? a (me e) 3 2 132 f 4 gm) 3:3 5:3 33% 5 gm) 8:2 3:2 15 0. 4 1. 6 1. 6 0.2 0. 4 0. 8 0. 2 0. 6 1. 6 0. 8 3. 2 0. 4 3. 2 0. 6 2.4 0. 2 0. 6 0.4 1. 2 0. 4 1. 6 0. 2 0. 4 0. 4 1. 6

In the above table INH refers to isoniazid, PAS to para-amino to pera-amlnobenzotc acid and PAS-amide to p-aminosallcylamid The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A therapeutic composition comprising, as its active ingredients, isoniazid and an acetylation inhibitor consisting of a soluble organic compound selected from compounds having the general formula:

where R is at least one member selected from the group consisting of hydrogen, halogen, hydroxyl, amino, lower alkyl radicals having not more than three carbon atoms,

phenyl and carboxyl, and where A is selected from the group consisting of pyridine, benzamide, benzoic acid :hydrazide, and benzoylhydroxamic acid, and pharmaceu- 55 tically acceptable salts thereof.

2. A therapeutic composition comprising as its active ingredients isoniazid and o-hydroxybenzalisonicotinyl hydrazone.

3. A therapeutic composition comprising as its active ingredients isoniazid and S-bromosalicylamide.

salicylic acid, PABA e References Cited in the file of this patent UNITED STATES PATENTS 2,721,827 Gustus Oct. 25, 1955 45 FOREIGN PATENTS 522,346 Belgium Sept. 15, 1953 OTHER REFERENCES 50 Thoren: Stanford Med. Bull., November 1952, pp. 

1. A THERAPEUTIC COMPOSITION COMPRISING, AS ITS ACTIVE INGREDIENTS, ISONIAZID AND AN ACETYLATION INHIBITOR CONSISTING OF A SOLUBLE ORGANIC COMPOUND SELECTED FROM COMPOUNDS HAVING THE GENERAL FORMULA: 